Methods and compositions to regulate plant transformation susceptibility

ABSTRACT

A genetic screen for Arabidopsis mutants displaying a hyper-susceptible to Agrobacterium transformation (hat) phenotype was performed. The gene disrupted in the hat3 mutant encodes a putative myb-family transcription factor (MTF) that negatively regulates susceptibility to Agrobacterium-mediated transformation. Over-expression of an integrin-like protein results in plants that are hyper-susceptible to transformation. Manipulation of MTF, members of this protein family, and members of the integrin domain-like protein family for example At14a allows improved control of Agrobacterium transformation, including in crops.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of co-pending U.S. application Ser. No.13/884,480, filed May 9, 2013, which is a U.S. nationalization under 35U.S.C. § 371 of International Application No. PCT/US2011/059944, filedNov. 9, 2011, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. provisional application No. 61/412,684, filed Nov. 11,2010, and 61/552,127, filed Oct. 27, 2011. The disclosures of thereferenced applications are incorporated herein by reference in theirentireties.

This invention was made with government support under Grant No.13-CPBR-3-0149 awarded by the United States Department of Agriculture.The United States Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 14, 2016, isnamed 246654_SEQ_ST25.txt and is 44,505 bytes in size.

BACKGROUND

A myb transcription factor designated MTF is disclosed that negativelyregulates plant transformation susceptibility. An integrin domain-likeprotein (which is under negative regulation by MTF) is involved inAgrobacterium attachment to plant cells and, thus, is a positivemediator of transformation: plants over-expressing the integrindomain-like protein are more susceptible to transformation, whereasplants mutant for the integrin domain-like protein are less susceptible.Manipulation of these elements allows improved control of Agrobacterialtransformation of plants, including in crops.

Agrobacterium-mediated plant transformation forms the basis for themodern agricultural biotechnology industry.

Agrobacterium tumefaciens causes the disease crown gall and geneticallytransforms numerous plant, fungal and animal species. VirulentAgrobacteria harbor a tumor-inducing (Ti) plasmid containing virulence(vir) genes required by the pathogen for transport of transferred (T-)DNA and virulence effector proteins to host cells. Induction of virgenes, processing of T-DNA from the Ti-plasmid, attachment of thebacteria to plants, and subsequent transfer of T-DNA and Vir proteins tohost cells are complex processes. Numerous studies have elucidated theevents governing these processes in the bacterium, but relatively littleis known about the plant contribution to transformation.

Although Agrobacterium has a broad host range, many economicallyimportant plants remain recalcitrant to transformation. Scientists haveused a variety of techniques to identify plant genes that are involvedin Agrobacterium-mediated transformation. Among these, forward andreverse genetic screens revealed more than 125 Arabidopsis and tobaccogenes involved in transformation. Collectively these lines, designated“rat” (resistant to Agrobacterium transformation), reflected theirattenuated response to transformation. The genes identified representsteps necessary for successful transformation, including bacterialattachment/biofilm formation, T-DNA and Vir protein transfer,cytoplasmic trafficking and nuclear targeting of the Vir protein/T-DNAcomplex (T-complex), Vir protein removal, T-DNA integration, andtransgene expression. However, none of these mutants identify genesglobally affecting plant transformation susceptibility.

SUMMARY

A myb transcription factor designated MTF is disclosed that negativelyregulates plant transformation susceptibility. An integrin domain-likeprotein (which is under negative regulation by MTF) is involved inAgrobacterium attachment to plant cells and, thus, is a positivemediator of transformation: plants over-expressing the integrindomain-like protein are more susceptible to transformation, whereasplants mutant for the integrin domain-like protein are less susceptible.Manipulation of these elements allows improved control of Agrobacterialtransformation of plants, including crops.

Agrobacterium-mediated transformation is the most widely used techniquefor generating transgenic plants. However, transformation remains amajor limitation to enhancement of major crops through biotechnology.The first known regulator of plant transformation susceptibility isdescribed herein. An Arabidopsis myb transcription factor (MTF)negatively regulates plant transformation susceptibility. DNA insertionsin the mtf gene made Arabidopsis lines hyper-susceptible totransformation by several Agrobacterium strains. In addition, RNAitargeting of MTF in the transformation-recalcitrant Arabidopsis ecotypeBl-1 resulted in increased transformation susceptibility accompanied byincreased bacterial attachment to roots.

Transcriptional profiling of wild-type and mtf mutant plants revealeddown-regulation of the WRKY48 transcription factor gene in the mtfmutants. Mutation of WRKY48 resulted in hyper-susceptibility totransformation, as did over-expression of two genes that wereup-regulated in the mtf mutants [At1g50060 or At5g15725]. Arabidopsisroots inoculated with Agrobacteria expressing a trans-zeatin secretion(TZS) gene showed decreased expression of MTF and a correspondingincrease in transformation susceptibility.

When the Arabidopsis myb gene is overexpressed in Arabidopsis, theplants grow much larger, the roots are longer, and the leaves are darkergreen. This may be a useful agronomic trait if this is confirmed forcrop plants grown in the field.

An integrin domain-like protein (which is under negative regulation byMTF) is involved in Agrobacterium attachment to plant cells and, thus,is a positive mediator of transformation: plants over-expressing theintegrin domain-like protein are more susceptible to transformation,whereas plants mutant for the integrin domain-like protein are lesssusceptible. Over-expression of the At14a gene produces an integrindomain-like protein in the Arabidopsis ecotype BI-1 increased bacterialbinding to roots, and also increases root transformation. This ecotypeis highly recalcitrant to Agrobacterium-mediated transformation, andbinds bacteria poorly to roots.

Increasing Agrobacterium-mediated transformation of recalcitrantspecies, and tissues of these species, is achieved by over-expressing ofthe At14a gene. In particular, some tissues that are easy to regeneratebut difficult to transform may not bind Agrobacterium well, andover-expressing At14a may improve binding and transformation.

Myb transcription factors and integrin-like proteins, alone or incombination are useful, to achieve a desired effect on transformation bymanipulating Agrobacterial transformation in a plant. For example, theintegrin-like protein is designated At14a, and the myb transcriptionfactor is MTF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I. Expression of MTF influences plant susceptibility toAgrobacterium-mediated transformation. 1A Percentage of root segmentsdeveloping tumors in plants inoculated with A. tumefaciens A208. 1BRepresentative plates showing increased transformation susceptibility ofmutants. 1C Map of T-DNA insertion positions in MTF. Numbers indicatenucleotide positions; +1 indicates translation start site 1D RelativeMTF transcript levels in wild-type, hat3, and mtf1-4 (previously mtf2)roots 1E Transformation susceptibility of root segments from wild-type,mtf1-4, and mtf1-4 plants complemented with a MTF cDNA. Numbers indicateindividual T2 generation lines 1F Relative MTF transcript levels inroots of wild-type, mtf1-4 (previously mtf2), and complemented mtf1-4(previously mtf2) lines 1, 2, 3, and 5 as in 1E 1G-1I, Down-regulationof MTF by RNAi in roots of ecotype Bl-1 increases transformationsusceptibility 1G and attachment of Agrobacteria to roots 1I. Numbersindicate individual T2 generation MTF-RNAi lines and empty vector (EV)line. 1H Relative MTF transcript levels in roots of Bl-1 and lines 2, 9,10, and EV. 1I Attachment of GFP-tagged A. tumefaciens A208 to rootsegments of Col-0, Bl-1 and MTF-RNAi lines 2, 8, 9, 10, and EV. Errorbars in all figures indicate s.e.m. from 3 (for relative transcriptlevels) or 5 (for percentage of roots developing tumors) replicates.

FIGS. 2A-2C. Phytohormone pre-treatment of Arabidopsis roots increasessusceptibility to Agrobacterium-mediated transformation. 2ARepresentative plates showing tumors on root segments from Arabidopsisecotypes following 0, 1, and 3 days of phytohormone pre-treatment beforeinfection with A. tumefaciens A208. 2B Percentage of root segmentsdeveloping tumors. 2C Transient transformation after 3 d phytohormonepre-treatment of root segments followed by infection with A. tumefaciensAt849.

FIGS. 3A-3D. A. tumefaciens tzs mutant is less virulent than thewild-type strain. 3A Percentage of root segments developing tumors afterinoculation with tzs mutant and wild-type A. tumefaciens. 3B RelativeMTF transcript levels in roots infected with tzs mutant and wild-type A.tumefaciens. 3C MTF promoter-EYFP construction expresses constitutivelyin transgenic Arabidopsis. 3D Inoculation of MTF promoter-EYFPtransgenic roots with TZS⁺ and tzs mutant A. tumefaciens.

FIGS. 4A-4E. Trans-zeatin treatment increases susceptibility toAgrobacterium-mediated transformation by strains lacking TZS. 4A-4BPercentage of root segments developing tumors in Col-0 4A and Bl-1 4Binoculated with A. tumefaciens A348 and A281 in the absence or presenceof trans-zeatin. Relative MTF transcript levels in root segments ofCol-0 4C and Bl-1 4D treated for two days with trans-zeatin. 4EAttachment of GFP-tagged A. tumefaciens A281 to root segments of Col-0and Bl-1 treated for 24 h with 0 or 1.4 μM trans-zeatin.

FIGS. 5A-5D. Manipulation of Arabidopsis genes that are regulated by MTFincreases susceptibility to Agrobacterium-mediated transformation. 5APercentage of root segments developing tumors in transgenic plantsover-expressing At1g50060 or At5g15725 cDNAs inoculated with A.tumefaciens A208. Numbers indicate individual T2 generation lines.Relative transcript levels of At1g50060 5B and At5g15725 5C afterinoculation with A. tumefaciens A208 (TZS⁺), A348 (TZS⁻), or A281(TZS⁻). 5D Percentage of root segments developing tumors inT-DNA-disruption mutants of genes down-regulated in mtf plants.

FIGS. 6A-6D. Arabidopsis mtf mutants are resistant to Botrytis cinerea.Col-0 and mtf1-4 (previously mtf2) plants were spray 6A ordrop-inoculated 6B with B. cinerea spores. Average lesion diameter 6Cwas calculated from drop-inoculated leaves 4 days post-inoculation. 6DRelative ORA59 transcript levels in Col-0 and mtf1-4 (previously mtf2)leaves 0, 24, and 48 h post-inoculation.

FIG. 7. Mutation of MTF increases root transformation susceptibility tomultiple Agrobacterium strains. Root segments from wild-type orhomozygous mtf1-4 (previously mtf2) mutant plants were inoculated withA. tumefaciens A348 or A281. The percentage of root segments thatdeveloped tumors was calculated. Error bars indicate s.e.m. from fivereplicates.

FIG. 8. Decreasing MTF expression increases transformationsusceptibility of Arabidopsis ecotype Bl-1. Transgenic T1 generationArabidopsis ecotype Bl-1 plants expressing a RNAi construction whichtargets MTF were inoculated with A. tumefaciens A208. The percentage ofroot segments that developed tumors was calculated. Numbers below thebars indicate individual Bl-1::MTF-RNAi lines. Error bar indicatess.e.m. from five replicates.

FIG. 9. RT-PCR analysis of transcripts of genes up-regulated in hat3 andhomozygous mtf1-4 (previously mtf2) roots. Amplified fragments werefractionated by electrophoresis through agarose gels, stained withethidium bromide, and photographed. The ACT2 gene was used as anormalization control.

FIG. 10. RT-PCR analysis of transcripts of genes down-regulated in hat3and homozygous mtf1-4 (previously mtf2) roots. Amplified fragments werefractionated by electrophoresis through agarose gels, stained withethidium bromide, and photographed. The ACT2 gene was used as anormalization control.

FIG. 11. Over-expression of several Arabidopsis genes that are regulatedby MTF increases plant susceptibility to Agrobacterium-mediatedtransformation. Root segments from T1 generation transgenic plantsover-expressing At2g40960, At1g50060, At5g46295, or At5g15725 cDNAs wereinoculated with A. tumefaciens A208. The percentage of root segmentsthat developed tumors was calculated. Numbers indicate individualtransgenic lines. Error bar indicates s.e.m. from five replicates.

FIGS. 12A-12B. Homozygous mtf1-4 (previously mtf-2) plants show noalteration in susceptibility to Alternaia brassicicola or Pseudomonassyringae DC3000. 12A Leaves of wild-type and homozygous mtf1-4(previously mtf2) mutant plants were inoculated with 5 μL of a 500,000spores/mL A. brassicicola spore suspension. The leaves were photographed5 d after inoculation. 12B Leaves of wild-type and homozygous mtf1-4(previously mtf2) mutant plants were inoculated with wild-type and hrcCmutant Pseudomonas syringae pv. tomato. After 0 and 3 d, leaf sectionswere ground and the bacteria plated.

FIG. 13. MTF-RNAi lines in Arabidopsis ecotype BI-1 show varying levelsof MTF transcripts.

FIG. 14. Decreasing MTF transcripts in the transformation-recalcitrantArabidopsis ecotype BI-1 increases susceptibility to Agrobacterium.Bacterial concentration (10⁸ cfu/mL).

FIG. 15. MTF-RNAi lines show increased attachment of GFP-labeledAgrobacteria.

FIG. 16. Arabidopsis and crop myb transcription factors are highlyhomologous. FIG. 16 discloses SEQ ID NOS 71-75, respectively, in orderof appearance.

FIG. 17. Expression of the rice MTF ortholog in the Arabidopsis mtf1-4(previously mtf2) mutant results in lower transformation susceptibility.

FIG. 18. A rice MTF-RNAi line shows increased transient transformation.

FIGS. 19A-19B. Expression of the Brassica oleracea MTF ortholog in theArabidopsis mtf1-4 (previously mtf2) mutant results in lowertransformation susceptibility. 19A shows results of a transient GUSassay; 19B a root tumorigenesis assay.

FIGS. 20A-20B. Expression of the Brassica napus MTF ortholog in theArabidopsis mtf1-4 (previously mtf2) mutant results in lowertransformation susceptibility. 20A shows results of a transient GUSassay; 20B a root tumorigenesis assay.

FIG. 21. Expression of the Brassica rapa MTF ortholog in the Arabidopsismtf1-4 (previously mtf2) mutant results in lower transformationsusceptibility. (Transient GUS assay).

FIGS. 22A-22E. MTF Sequences: Double underlined nucleotides indicatestart codons; single underlined nucleotides indicate stop codons; italicbold nucleotides indicate part of the 5′- and 3′ untranslated sequenceson the cDNA clones: 22A Arabidopsis MTF and MTF (SEQ ID NOS 76 and 74,respectively, in order of appearance); 22B-22E orthology sequences[Rice, Brassica napus, Brassica rapa, Brassica oleracea] (SEQ ID NOS 77,75; 78, 71; 79, residues 1-233 of SEQ ID NO: 72; 80 and 73,respectively, in order of appearance).

FIG. 23. At14a: The mtf1-4 (previously mtf2) mutant shows increasedAt14a transcript levels; At14a was of interest because its expression isup-regulated in the Arabidopsis mtf myb transcription factor mutant;this mutant is hyper-susceptible to Agrobacterium-mediatedtransformation.

FIG. 24. At14a Sequences: The Arabidopsis data bases indicate that thereare two identical At14a gene sequences (“At3G28290” and “At3G28300” bothdisclosed as SEQ ID NO: 81), plus two related sequences (SEQ ID NOS82-83, respectively, in order of appearance).

FIGS. 25A-25B. At14a Transformation: The transformation susceptibilityof the Arabidopsis At14a mutant is lower than that of wild-type Col-0plants. 25A shows a transient GUS assay; 25B antibiotic resistant calli.

FIG. 26. At14a Binding: Arabidopsis At14a shows decreased binding ofGFP-labeled A. tumefaciens A348.

FIG. 27. At14a Binding: Arabidopsis At14a mutant shows decreased bindingof GFP-labeled A. tumefaciens A208.

FIG. 28. At14a and mtf1-4: The At14a mutant shows decreased binding, andthe mtf1-4 (previously mtf2) shows increased binding, of A. tumefacienscompared to Arabidopsis Col-0 (using scanning electron microscopy ofunlabeled Agrobacteria).

FIG. 29. Arabidopsis plants infected with TZS and/or iP-producing A.tumefaciens strains show lower amounts of MTF transcripts.

FIGS. 30A-30C. Arabidopsis plants infected with an A. tumefaciens miaAmutant show decreased transformation susceptibility. 30A binding; 30Bshows a transient GUS assay; 30C shows kanamycin resistant calli.

DETAILED DESCRIPTION

A genetic screen for Arabidopsis mutants displaying a hyper-susceptibleto Agrobacterium transformation (hat) phenotype was performed. The genedisrupted in the hat3 mutant encodes a putative myb-family transcriptionfactor (MTF) that negatively regulates susceptibility toAgrobacterium-mediated transformation.

Identification and Characterization of Mtf Mutants

To identify mutants with increased susceptibility toAgrobacterium-mediated transformation, ˜4000 mutagenized plants werescreened from an Arabidopsis T-DNA activation-tagged library (Weigel,2000). The mutant hat3 displayed a ˜10-fold increase in transformationsusceptibility (FIG. 1A, 1B). TAIL-PCR (Liu et al., 1995) was used toidentify the T-DNA/plant junction in hat3, and it was discovered thatthe T-DNA had inserted into the 5′ untranslated region of a putative mybtranscription factor (MTF) gene, At2g40970, 36 bp upstream of the startcodon (FIG. 1C). MTF has a single Myb DNA-binding domain of the SHAQKYF(SEQ ID NO: 1) type that is unique to plants, and is a member of afive-gene family (Hazen et al., 2005). The DNA-binding domain is similarto those found in proteins associated with two-component signaltransduction systems (Hwang et al., 2002), the B-type Arabidopsisresponse regulators (ARRs), GOLDEN2-LIKE (GLK), and PRR2 (Hazen et al,2005).

Homozygous mutant plants were not recoverable from self-fertilizedprogeny of hat3, suggesting that complete disruption of MTF may belethal. Self-fertilization of three additional T-DNA MTF insertionmutants, SALK_072082 (mtf1), SALK_072083 (mtf1-4), and SALK_102624(mtf3), resulted in a homozygous mutant only for mtf1-4 (previouslymtf2). The insertion in mtf1-4 (previously mtf2) permitted expression of˜85% of the MTF open reading frame, indicating that the majority of MTFprotein is essential for Arabidopsis viability. Homozygous mtf1-4(previously mtf2) plants showed an ˜11-fold increase in transformationsusceptibility. Heterozygous mtf1 and mtf3 mutants displayed 4-7-foldincreased transformation susceptibility (FIG. 1A, 1B). Thus, all fourmtf mutant lines displayed a hat phenotype, highlighting the importanceof MTF in transformation. Quantitative real-time RT-PCR assays revealedthat MTF transcript levels decreased 2-fold in mtf1-4 (previously mtf2)and >12-fold in hat3 (FIG. 1D), demonstrating that transformationsusceptibility negatively correlates with MTF transcript levels.

The transformation experiments described herein were carried out usingA. tumefaciens A208 that contains a nopaline-type of Ti plasmid.Commonly used Agrobacterium strains were, for example A208, A348, A281(Zhu et al., 2003; and Nam et al., 1999). To assess whether mtf1-4(previously mtf2) shows increased susceptibility to other A. tumefaciensstrains, root transformation assays were conducted using theoctopine-type strain A348 and the succinamopine-type strain A281. Themtf1-4 (previously mtf2) mutant displayed 2-3-fold increasedtransformation susceptibility to these strains (FIG. 7). Thus, MTF playsan important role in plant susceptibility to different Agrobacteriumstrains.

Further studies used homozygous mtf1-4 (previously mtf2) plants. Ectopicexpression of the MTF cDNA in mtf1-4 (previously mtf2) resulted inseveral transgenic lines with restored levels of wild-typesusceptibility to Agrobacterium-mediated transformation (FIG. 1E). Thesetransgenic lines individually expressed various levels of MTF mRNA (FIG.1F). Complementation experiments confirm that disruption of the MTF geneis responsible for increased transformation susceptibility.

The mtf1-4 (previously mtf2) mutant is hyper-susceptible to differentstrains of A. tumefaciens carrying nopaline-, octopine-, andsuccinomanopine-type Ti plasmids, indicating that MTF is a negativeregulator of Agrobacterium-mediated transformation. Transformationrecalcitrance of some Arabidopsis ecotypes results from decreasedbinding of Agrobacterium to roots. Other ecotypes are debilitated inT-DNA integration, a late stage of transformation (Nam et al., 1997).Reducing MTF expression in Bl-1, a highly recalcitrant ecotype,increased transformation susceptibility and bacterial attachment,highlighting the potential to increase transformation susceptibility ofrecalcitrant plant species by down-regulating expression of MTForthologs.

The importance of phytohormones in increasing transformation promptedinvestigation of the role of cytokinins in transformation. Agrobacteriumstrains containing nopaline-type Ti plasmids secrete trans-zeatin,mediated by the vir region-localized gene TZS. A. tumefaciens tzsmutants are less virulent than are TZS⁺ strains. The presence of TZS onthe bacterial surface (Aly et al, 2008) may mean that metabolites fromwounded plant cells may be converted into trans-zeatin at infectionsites, resulting in down-regulation of MTF and consequent increasedtransformation susceptibility. Indeed, exogenous application of kinetinduring infection increased the susceptibility of Arabidopsis rootsinfected with an Agrobacterium tzs mutant (Hwang et al., 2010).Down-regulation of MTF expression by cytokinins provides a molecularexplanation for the importance of TZS to Agrobacterium-mediatedtransformation (Zhan et al, 1990). Although influential, cytokininsignaling is not essential for Agrobacterium-mediated transformationbecause many virulent Agrobacterium strains do not secrete cytokinins.

Regulation of gene expression by MTF is highly specific. Fewer than 40genes are significantly up- or down-regulated ≥1.5-fold in the mtfmutants. One of the up-regulated genes, At1g50060 encoding a basicPR1-like protein, increased transformation susceptibility whenover-expressed in Arabidopsis. Unlike its acidic counterpart, PR-1,At1g50060 is not salicylic acid (SA)-responsive, pathogen-induced, noris its expression correlated with the establishment of systemic acquiredresistance (Niki et al., 1998). However, At1g50060 transcripts arenegatively regulated by a variety of biotic and abiotic stresses(Zimmerman et al., 2004). Thus, At1g50060 does not encode adefense-related protein. Increased transformation susceptibility of thewrky48 mutant suggests that Agrobacterium manipulates host defenseresponses to its advantage. Previously Veena et al. (2003) showed thatinfection of plant cells by transfer-competent Agrobacterium strainssuppresses host defense gene expression 30-36 h after infection,although these genes are induced as early as 3-12 h after infection(Veena et al., 2003). MTF is a specific regulator of plantsusceptibility to Agrobacterium as evidenced by lack of increasedsusceptibility to A. brassicicola and P. syringae. Increased resistanceof the mtf mutant to Botrytis is likely due to downstream responses todecreased MTF expression.

In conclusion, MTF was identified as the first known regulator of plantsusceptibility to Agrobacterium-mediated transformation. MTF regulatesat least three genes independently capable of increasing transformationsusceptibility. MTF also affects Agrobacterium binding to roots andintegrates cytokinin secretion by Agrobacterium with transformationsusceptibility. These findings pave the way for identifying orthologs ofMTF in transformation-recalcitrant plant species and manipulating thesegenes to increase transformation efficiency of economically importantcrops.

EXAMPLES

Examples are provided for illustrative purposes and are not intended tolimit the scope of the disclosure.

Example 1: Decreased MTF Expression in Arabidopsis Ecotypes IncreasesTransformation Susceptibility

The hat3 and mtf1-4 (previously mtf2) mutants are in the Columbiabackground, an ecotype relatively amenable to root transformation.Arabidopsis ecotype Bl-1 is highly recalcitrant to root transformation(Nam et al., 1997), but can be transformed using a floral dip method(Mysore et al., 2000). MTF genes of ecotypes Columbia and Bl-1 areidentical. An RNAi expression construction targeting MTF transcripts wasintroduced into ecotype Bl-1 and the derived transgenic lines werescreened for root transformation susceptibility. Eight of the 10 testedT1 generation transgenic plants exhibited increased susceptibility (FIG.8). 25 T2 generation plants from each of five MTF-RNAi lines weretested, along with a RNAi empty vector line. Three of these transgeniclines continued to show higher transformation susceptibility (FIG. 1G).RNAi lines 2 and 9, that had increased transformation susceptibility,showed 4.6- and 7-fold decreases in MTF transcripts, respectively,whereas line 10, that did not have altered susceptibility, showed only a2-fold decrease in MTF transcript levels (FIG. 1H). A transgenic linecontaining an empty RNAi vector did not display altered transformationsusceptibility or altered MTF transcript levels. These results indicatethat transformation susceptibility of Bl-1 plants is dependent on thelevel of MTF transcripts.

Earlier studies indicated that roots of ecotype Bl-1 do not bindAgrobacteria well. A. tumefaciens expressing GFP showed increasedbacterial attachment in the high-transforming transgenic Bl-1 RNAi lines2 and 9 compared to that of the low-transforming line 10, the empty RNAivector line, and wild-type Bl-1 (FIG. 1I), suggesting that decreased MTFtranscripts in Bl-1 increase susceptibility during the early attachmentstage of the transformation process.

Example 2: Phytohormone Treatment Increases TransformationSusceptibility

Chateau et al. (2000) reported that phytohormone preincubation ofArabidopsis petioles increases transformation susceptibility, andhormone pre-treatment is part of the protocol to generate transgenicArabidopsis plants from roots (Valvekens et al., 1988). Becausephytohormone pretreatment of Arabidopsis root segments may enhancetransformation susceptibility, which may be important in light of thefact that nopaline-type Agrobacterium strains express a trans-zeatinsecretion (TZS) gene, and thus secrete cytokinins into the medium.

Root segments from five transformation-recalcitrant Arabidopsis ecotypes(Bl-1, Bla-2, Cal-0, Dijon-G, and Petergof) and atransformation-susceptible ecotype (Ws-2) were incubated on callusinducing medium (CIM) containing phytohormones prior to infection byAgrobacterium and scored for transformation susceptibility. All ecotypesdisplayed increased transformation susceptibility after one day ofphytohormone pre-treatment (FIG. 2A, 2B). There was a further increasein transformation frequency after three days of phytohormonepre-treatment.

Whether phytohormone pre-treatment of Arabidopsis roots enhances thefrequency of transient transformation was investigated, a process thatdoes not require T-DNA integration into the plant genome.β-glucuronidase (GUS) activity, resulting from the transfer of agusA-intron gene from Agrobacterium to plants, is a standard assay fortransient transformation (Narasimhulu et al., 1996). Hormonepre-treatment of roots also increased transient transformation (FIG.2C). Petiole explants of Arabidopsis treated with phytohormones beforeAgrobacterium infection showed actively dividing and dedifferentiatedcells, and increased transformation efficiency. Increased DNAduplication and cell division of phytohormone treated Petunia hybridacells correlated with increased Agrobacterium-mediated transformation(Villemont et al., 1997). Thus, phytohormone treatment sensitizes rootsto Agrobacterium-mediated transformation at an early step (prior toT-DNA integration) of the transformation process.

Example 3: MTF Expression is Repressed by Cytokinins from Agrobacterium

Ti-plasmids of some nopaline-type Agrobacterium strains carry a TZS genethat directs synthesis and secretion of cytokinins (Regier et al., 1982;Beaty et al., 1986; and Powell et al., 1988). TZS promotestransformation both by nopaline-type A. tumefaciens strains and, whentransferred to strain 1855, A. rhizogenes strains. A. tumefaciensstrains harboring nopaline-type Ti plasmids secrete trans-zeatin ortrans-zeatin ribosides into the medium in amounts >1 μg/L (Claeys etal., 1978; McCloskey et al, 1980).

Tumorigenesis assays were conducted on Arabidopsis roots infected withthe TZS⁺ strain A. tumefaciens NT1RE(pJK270) and the tzs frameshiftmutant NT1RE(pJK270tzs-fs). Arabidopsis root segments infected with thetzs mutant developed fewer tumors than did roots infected with thewild-type strain (FIG. 3A). Root segments infected with wild-typebacteria had 10-fold fewer MTF transcripts than did roots infected withtzs-mutant bacteria (FIG. 3B). These results indicate that MTF isdown-regulated by trans-zeatin produced by A. tumefaciens, leading toaltered transformation susceptibility.

Example 4: TZS-Expressing Agrobacteria Repress Expression of MTF

Decreased MTF transcript levels in roots co-cultivated with TZS⁺ A.tumefaciens suggests an early involvement of trans-zeatin and MTF intransformation. To determine in which root tissues this decrease in MTFexpression was most pronounced, transgenic Arabidopsis lines expressingEYFP under control of the MTF promoter were generated. MTF promoteractivity was constitutive in all examined plant tissues (FIG. 3C). Thehighly-expressing line Col7-P_(MTF)-EYFP4 was used to assess whetherroot tissues exhibited altered MTF expression when infected with a TZS⁺A. tumefaciens strain. Fluorescence decreased in roots by 48 h ofco-cultivation, most noticeably in the epidermal and cortical cells ofthe elongation zone, the region most susceptible to transformation (FIG.3D). This decrease in fluorescence was not observed in roots incubatedwith the tzs frameshift mutant. These results are consistent with thedecreased MTF transcript levels observed in roots co-cultivated withTZS⁺ bacteria.

Example 5: MiaA-Expressing Agrobacteria Repress Expression of MTF andare More Susceptible to Transformation

MiaA encodes an tRNA-isopentenyltransferase that isopentenylates adenineresidues in tRNAs. Breakdown of tRNAs can release isopentenyladenine, acytokinin. When Arabidopsis root segments are inoculated withAgrobacteria that contain a wild-type MIAA gene, the accumulation of MTFtranscripts is repressed (FIG. 29). miaA mutant bacteria are lessvirulent than are wild-type bacteria (FIG. 30).

Example 6: Cytokinin Enhances Attachment of TZS-Lacking A. tumefaciensStrains

To determine whether exogenous application of trans-zeatin to rootscould influence transformation susceptibility, Arabidopsis roots wereincubated on medium containing trans-zeatin and they were infected withA. tumefaciens A348 or A281. Neither of these strains harbors TZS.Trans-zeatin concentrations representing the range secreted bynopaline-type A. tumefaciens strains were used. Trans-zeatin treatmentof Col-0 roots resulted in a 4-8-fold increase in transformationefficiency by these A. tumefaciens strains. Ecotype Bl-1 roots infectedwith these strains showed a 2-3-fold increase in susceptibility (FIG.4A, 4B). Incubation of roots on trans-zeatin decreased MTF transcriptlevels by 30-60% and also increased attachment of A. tumefaciens A281(FIG. 4C, 4D, 4E).

Example 7: Decreased MTF Expression Alters Expression of Genes Importantfor Agrobacterium-Mediated Transformation

The Arabidopsis ATH1 Genome Arrays were used to identify genes whoseexpression is altered in wild-type, heterozygous hat3, and homozygousmtf1-4 (previously mtf2) Arabidopsis roots. A total of 39 genesexhibited statistically significant differential expression between bothmtf mutants and the wild-type, and had a difference greater than1.5-fold (Table 1). Of these, 23 genes were commonly up-regulated and 16genes were commonly down-regulated in both mtf mutants compared to thewild-type. These results were validated using RT-PCR (FIGS. 9 and 10).cDNAs of four genes At2g40960, At1g50060, At5g46295, At5g15725 that wereup-regulated in both mtf mutants were overexpressed. Transgenic T1 linesover-expressing At2g40960 and At5g46295 did not exhibit a hat phenotype(FIG. 11). However, several T1 lines over-expressing At1g50060 andAt5g15725 showed increased transformation susceptibility that carriedover to the T2 generation (FIG. 5A). At1g50060 is a putativepathogenesis-related 1 (PR-1)-like protein proposed to be a serineprotease involved in various signaling processes (Fernandez et al, 1997;Milne et al., 2003). At1g50060 transcript levels in root segmentsinfected with A. tumefaciens A208 (TZS⁺) and strains A348 and A281 wereassessed (TZS⁻) and observed increased transcript levels only inA208-infected roots (FIG. 5B). Presumably, cytokinins produced by A.tumefaciens A208 regulate expression of MTF in the roots, which in turnregulate expression of At1g50060.

At5g15725 is annotated as an unknown protein (Tair;http://www.arabidopsis.org/). Arabidopsis root segments were infectedwith A. tumefaciens strains A348, A208, or A281. Expression of At5g15725was up-regulated by all three strains; however, the highest transcriptlevels were found after infection by the TZS-producing strain A208 (FIG.5C) which may be related to trans-zeatin production.

To assess the effect of genes down-regulated by MTF, roots ofindependent T-DNA insertion mutants in At5g49520 (wrky48), At3g56710(sigA), At4g25470 (dreb1c), At5g39670 (cbp1) and At2g43290 (mss3) wereassayed. The wrky48 mutant exhibited a mild hat phenotype (FIG. 5D).None of the other tested mutants displayed increased transformationsusceptibility. WRKY48 is a transcriptional activator that repressesplant basal defenses (Xing et al., 2008). Results indicate that defensegenes regulated by WRKY48 do not play a major role in protecting thehost from Agrobacterium infection, or that Agrobacterium somehow targetsand/or recruits host defenses to its advantage.

Example 8: Effect of the MTF Mutation on Infection by OtherPhytopathogens

A question was whether mtf1-4 (previously mtf2) plants showed alteredsusceptibility to other pathogens. Col-0 and mtf1-4 (previously mtf2)plants showed similar symptoms when infected with the necrotrophicfungus Alternaria brassicicola, and the virulent DC3000 or thenon-pathogenic hrcC⁻ mutant strain of Pseudomonas syringae pv. tomato(FIG. 12). However, mtf1-4 (previously mtf2) plants showed increasedresistance to infection by Botrytis cinerea (FIG. 6A). Leaves of mtf1-4(previously mtf2) drop-inoculated with B. cinerea displayed smallerlesions than did wild-type plants (FIGS. 6B, 6C). Resistance tonecrotrophic pathogens is mediated through jasmonic acid (JA) andethylene. Microarray data revealed that At1g06160 (ORA59), encoding anoctadecanoid-responsive Arabidopsis AP2/ERF transcription factor, issignificantly up-regulated (1.6-fold; p<0.0001) in the mtf1-4(previously mtf2) mutant. Because B. cinerea infection down-regulatesMTF³⁵, ORA59 transcript levels were quantified in leaves of mtf1-4(previously mtf2) and wild-type plants 0, 24, and 48 hourspost-inoculation (hpi) with B. cinerea spores. By 24 hpi, more than a3-fold increase in ORA59 transcript levels was seen in mtf1-4(previously mtf2) compared to infected wild-type plants (FIG. 6D).Constitutive over-expression of ERF1 induces the expression of thedefense-response genes PDF1.2 and ChiB (PR-3), and confers resistance toB. cinerea (Berrocal-Lobo et al., 2002). Thus, the modestly higherlevels of ChiB (1.3-fold; p=0.004), and B. cinerea-induced up-regulationof ORA59 in mtf1-4 (previously mtf2), likely contribute to increasedresistance to B. cinerea.

Example 9: Manipulation of Myb Transcription Factors to Improve CropTransformation

An Arabidopsis myb transcription factor (MTF) was identified which is anegative regulator of plant susceptibility to Agrobacterium-mediatedtransformation. Decreased expression of MTF results in a 10- to 15-foldincrease in transformation frequency of the Arabidopsis ecotype Columbia(Col). Increased transformation susceptibility correlates with anincrease in binding of Agrobacteria to the plant surface. This bindingis mediated by an integrin-like protein. MTF expression is negativelyregulated by cytokinins secreted by Agrobacterium cells, mediated bymiaA and/or tzs.

mtf RNAi plants were generated in the transformation-recalcitrantecotype BI-1 and transformation susceptibility was determined.

MTF orthologs were identified from crop species.

Using a bioinformatic approach i.e. “masking” the central myb DNAbinding domain of MTF, and searching for proteins homologous to the N-and C-terminal regions of MTF, the correct myb orthology was verified byintroducing the cDNA of an ortholog into the Arabidopsis mtf1-4(previously mtf2) mutant and assaying for decreased transformationsusceptibility.

MTF ortholog expression is identified in crop species using RNAi (orTILLING (Targeting Induced Local Lesion in Genomes)) and testingtransformation susceptibility.

Results showed the following:

1. Decreased expression of MTF in A. thaliana ecotype Bl-1 results inincreased Agrobacterium attachment and transformation susceptibility.

2. MTF orthologs were identified from rice and three Brassica species.The identity of these orthologs was confirmed by functionalcomplementation of the Arabidopsis mtf1-4 (previously mtf2) mutant.

3. Decreased expression of the rice MTF ortholog by RNAi results inincreased rice transformation susceptibility.

Expression of the Brassica MTF orthologs are determined in their nativespecies and the resulting plants are assayed for increasedtransformation susceptibility.

Expression of the rice MTF ortholog is decreased intransformation-recalcitrant japonica and indicia lines and the resultingplants are assayed for increased transformation susceptibility.

A transient RNAi system, delivered by Agrobacterium, silences crop MTForthologs while simultaneously delivering genes of interest to thesespecies.

MTF orthologs from soybean and wheat were identified and are silenced.Putative orthologs were identified using bioinformatics. (using BLAST®(Basic Local Alignment Search Tool))

Example 10: Involvement of the Integrin Domain-Like Protein At14a inAgrobacterium-Mediated Transformation. (See FIGS. 23, 24, 25A, 25B, 26,27, 28)

Over-expression of the At14a gene in the Arabidopsis ecotype BI-1increased bacterial binding to roots, and also increases roottransformation. This ecotype is highly recalcitrant toAgrobacterium-mediated transformation, and binds bacteria poorly toroots.

Increasing Agrobacterium-mediated transformation of recalcitrantspecies, and tissues of these species, is achieved by over-expressing ofthe At14a gene. In particular, some tissues that are easy to regeneratebut difficult to transform may not bind Agrobacterium well, andover-expressing At14a may improve binding and transformation.

MATERIALS AND METHODS

A. tumefaciens was cultured in Yeast Extract-Peptone medium(Lichtenstein et al., 1986) containing the appropriate antibiotics. Roottransformation assays were carried out as previously described by Nam etal. with minor modifications (Tenea et al., 2009). MS basal mediumlacking phytohormones was used to select for tumors. GUS activity assayswere carried out after infection of root segments with A. tumefaciensAt849 (Narasimhulu et al., 1996) for 4-6 d, using X-gluc (Jefferson etal., 1987). Detailed procedures for identifying and screeningArabidopsis mutants, generating transgenic plants, quantitativereal-time RT-PCR, bacterial attachment assays, phytohormone treatment ofplant roots, microarray experiments, and infection of plants withpathogenic microbes are available in the Methods.

Agrobacterium Culture, Plant Growth Conditions and Transformation Assays

A. tumefaciens was cultured in Yeast Extract-Peptone medium containingappropriate antibiotics. Root transformation assays were carried out aspreviously described with minor modifications. MS basal medium lackingphytohormones was used to select for tumors. GUS activity assays werecarried out after infection of root segments with A. tumefaciens At849for 4-6 d, using X-gluc.

Arabidopsis Mutants

˜4000 mutagenized plants from an activation-tagged library were screenedat low Agrobacterium inoculation densities (10⁵ and 10⁶ cfu/mL) forincreased root transformation. TAIL-PCR was utilized to identify theT-DNA/plant junction from hat3. Primers for TAIL-PCR are listed in Table2.

Seeds of the T-DNA insertion MTF mutants SALK_072082 (mtf1), SALK_072083(mtf1-4) (previously mtf2), and SALK_102624 (mtf3) (Alonso et al., 2003)were obtained from the Arabidopsis Biological Resource Center (Columbus,Ohio). The mutants were genotyped using primers listed in Table 2.

Generation of Transgenic MTF-Complemented Plants

MTF cDNA was synthesized from 1-2 μg RNA using oligo(dT) and theSuperscriptIII First Strand Synthesis System for RT-PCR™ (Invitrogen,Carlsbad, Calif.), following the manufacturer's protocol. Primersequences are listed in Table 2. The polymerase chain reaction (PCR) wasconducted using PfuTurbo DNA polymerase (Stratagene, La Jolla, Calif.)and 200 ng of Arabidopsis Columbia root cDNA. PCR products were clonedinto the SmaI site of pBluescript II SK+ (Stratagene). MTF cDNA wasexcised using XhoI and SpeI and cloned into the binary vector pE1775(Lee et al., 2007). The resulting construction, pE3263, was introducedinto A. tumefaciens GV3101 by electroporation and used for floral diptransformation (Clough and Bent, 1998) of the mutant mtf1-4. Transgenicplants were selected on B5 medium containing 20 μg/mL hygromycin.

Quantitative Real-Time RT-PCR Analysis

Real-time RT-PCR was carried out using total RNA isolated in triplicatefrom roots of plants grown in liquid B5 medium. PCR was performed intriplicate on an ABI Prism 7500 Sequence Detection System (AppliedBiosystems, Foster City, Calif.). Expression levels were calculated bythe relative standard curve method (Applied Biosystems) for alltranscripts except ORA59, where the comparative cycle threshold method(Applied Biosystems) was used, and normalized to Actin2 transcriptlevels. Transcript levels of genes identified in microarray experimentswere validated by RT-PCR. The list of primers is given in Table 2.

Generation of MTF-RNAi Lines

MTF-RNAi lines were generated using pFGC1008 (GenBank AccessionAY310333). The RNAi construct (pE3387) contained a ˜400 bp cDNA fragmentof MTF amplified using primers listed in Table 2. The MTF fragment wasoriented as an inverted repeat with each repeat separated by a fragmentfrom the gusA gene. RNAi lines, in ecotype Bl-1, were produced byfloral-dip transformation using A. tumefaciens GV3101. Transgenic plantswere selected on B5 medium containing hygromycin.

Bacterial Attachment Assays

Root segments of Arabidopsis Bl-1 and MTF-RNAi lines were incubated withA. tumefaciens A208 containing pJZ383 (P_(tac)::GFP). Root segments wereco-cultivated with 10⁵ cfu/mL (ecotype Columbia) or 10⁸ cfu/mL (ecotypeBl-1) for 24 h in B5 minimal medium. Root segments were rinsed andvisualized by epifluorescence microscopy.

Generation of MTF Promoter-EYFP Transgenic Plants

˜1.2 kb of the MTF promoter was amplified using a forward primerincorporating an AgeI restriction site at the 5′ end and a reverseprimer incorporating the sequence for the first ten amino acids of MTFand a BamHI restriction site. Primers are listed in Table 2. Theamplification product was cloned into the SmaI site of pBluescriptIISK+. The MTF promoter was excised using AgeI and BamHI and cloned intothese sites of pSAT6-EYFP-N1⁴⁶ as a translational fusion with EYFP. Theexpression cassette was cloned as a PI-PspI fragment into pPZP-RCS2(Tzfira et al., 2005). The resulting plasmid was transformed into A.tumefaciens GV3101 and used for floral-dip transformation of ArabidopsisCol-0. Transgenic plants were selected on B5 medium supplemented withhygromycin.

Phytohormone Treatment of Plant Roots

Plants of Arabidopsis ecotypes Ws-2, Bl-1, Bla-2, Cal-0, Dijon-G, andPetergof were grown as described by Nam et al. and roots were excisedand incubated on CIM for 0, 1, or 3 days prior to cutting into segmentsand infection with A. tumefaciens A208 for tumorigenesis assays orstrain At849 for transient GUS expression assays.

For assessing the effect of cytokinins on MTF transcript levels andtransformation, root segments from Arabidopsis Col-0 or Bl-1 wereincubated on MS medium supplemented with 0, 1.4 or 14 μM trans-zeatin,and co-cultivated with either A. tumefaciens A348 or A281 for 48 h.Roots were infected with bacteria at 10⁶ cfu/mL (Col-0) or 10⁸ cfu/mL(Bl-1). Following infection, root segments were either transferred to MSbasal medium containing 100 μg/mL Timentin and incubated for 4-5 weeksbefore recording the percentage of root segments developing tumors, orused for RNA isolation.

Agrobacterium attachment assays were conducted as described herein.Col-0 and Bl-1 root segments were co-cultivated with A281 at 10⁶ or 10⁸cfu/mL, respectively, for 24 h in the presence or absence of 1.4 μMtrans-zeatin.

Microarray Analysis

Surface-sterilized seeds of wild-type, hat3, and mtf1-4 (previouslymtf2) were germinated in B5 medium and seedlings grown for 2-weeks at23° C. under a 16 h light/8 h dark photoperiod. Three biologicalreplicates, each consisting of twenty seedlings of each line transferredto liquid B5 medium, were grown for 12 days. Roots were frozen in liquidN₂. RNA was isolated using Trizol reagent (Invitrogen). Microarrayexperiments were performed according to the Affymetrix GeneChipExpression Analysis Manual (http://www.affymetrix.com) using ArabidopsisATH1 Genome Arrays (Affymetrix) at the Purdue University GenomicsCenter. GeneChip operating software was used to produce CEL filescontaining raw probe intensities for the arrays. Data from these fileswere read with “Biobase” and “affy” packages in R/Bioconductor(Gentleman et al., 2004) for analysis of genomic data. A backgroundcorrection was performed on the perfect match intensities to makesignals from different chips comparable. A robust local regression wasemployed to normalize background corrected data. An analysis of variance(ANOVA) method was employed as previously described by Chu et al., 2002,to detect probe sets which are differentially expressed between twolines using the natural log of the background corrected, normalized dataas the gene expression level. To determine whether there was astatistically significant difference between two lines, it wassufficient to test whether the line effect was different from zero. ThisANOVA model was performed for Col vs hat3, Col vs mtf1-4 (previouslymtf2), and mtf1-4 vs hat3. Both the false discovery rate (FDR) approach(Benjamini et al., 1995) and Holm's sequential Bonferroni correctionprocedure (Holm, 1979) were used to adjust for multiple testing, with asignificance level a of 0.05.

Generation of Transgenic Arabidopsis Lines Over-Expressing GenesUp-Regulated in mtf Mutants

cDNAs of At2g40960, At1g50060, At5g46295, and At5g15725 were amplifiedusing primers containing KpnI and SacI sites, and cloned into the SmaIsite of pBluescriptII SK+. The primers used for amplification are listedin Table 2. DNA was digested with KpnI and SacI and cloned into pE1775(Lee et al., 2007). The resulting constructs were introduced into A.tumefaciens GV3101 by electroporation and used for floral-diptransformation of Arabidopsis Col-0. Transgenic plants were selected onB5 medium supplemented with hygromycin.

Disease Assays on Col-0 and Mtf1-4 (Previously Mtf2)

Fungal and bacterial cultures were maintained and disease assaysperformed as previously described by Mengiste et al., 2003. Botrytiscinerea strain B05-10 spores were harvested 10 days after initiatingculture and re-suspended in 1% Sabouraud Maltose Broth (SMB) media(DIFCO, Sparks, Md.) at a concentration of 2.5×10⁵ spores/mL for spray-and drop-inoculation of whole plants. Alternaria brassicicola sporeswere harvested and re-suspended in distilled water at a concentration of5×10⁵ spores/ml for drop-inoculation of detached leaves. Disease assayswith Pseudomonas syringae pv. tomato DC3000 and hrcC were done asdescribed.

TABLE 1 Fold-change of significantly differentially regulated genes intwo MTF mutants compared to the wild-type, identified by microarrayanalyses Fold change Gene Annotation hat3 mtf1-4 Up-regulated genesAt1g71870 MATE efflux family protein 3.9 3.3 At3g05730 defensin-like(DEFL) family protein 3.0 3.1 At2g25510 unknown protein 1.6 2.6At3g16670 phylloplanin precursor (T-phylloplanin) 2.4 2.4 At5g10040hypothetical protein 2.6 2.1 At2g02990 ribonuclease, RNS1 2.2 2.0At2g41230 similar to ARL (ARGOS-LIKE) 1.4 2.0 At2g40960 nucleic acidbinding 1.5 1.9 At1g50060 putative pathogenesis-related protein 1.3 1.8At5g46295 expressed protein 1.8 1.7 At5g05900 UGT 76C3 1.3 1.7 At3g62760glutathione transferase III-like protein 1.4 1.7 At5g14750 mybtranscription factor werewolf 1.5 1.7 (WER)/MYB66 At5g15725 expressedprotein 1.3 1.6 At1g74490 putative protein kinase 1.6 1.6 At4g38080putative hydroxyproline-rich glycoprotein 1.9 1.6 family proteinAt4g29690 nucleotide pyrophosphatase-like protein 1.9 1.6 At2g25980jacalin lectin family protein 1.4 1.5 At1g74500 putative DNA-bindingbHLH protein 1.4 1.5 At1g23160 GH3-like auxin-regulated protein 1.7 1.5At5g44260 zinc finger (CCCH-type) family protein 1.3 1.5 At2g40010 60Sacidic ribosomal protein P0 1.4 1.5 At3g17990 phosphoethanolamine 1.51.5 N-methyltransferase 1 Down-regulated genes At2g40970 myb familytranscription factor 4.0 4.3 At1g35210 expressed protein 1.4 2.3At1g77640 ERF/AP2 transcription factor DREBA5 1.7 2.2 At3g56710 SigAbinding protein 1.3 1.9 At5g37770 calmodulin-related protein 2, 1.3 1.8touch-induced (TCH2) At5g39670 calcium-binding protein (CBP1) 1.5 1.8At2g43290 calmodulin-like protein (MSS3) 1.3 1.8 At4g25470 DRECRT-binding protein DREB1C 1.4 1.7 At5g49520 WRKY48 1.3 1.7 At4g11280ACC synthase (AtACS-6) 1.3 1.6 At1g51920 expressed protein 1.4 1.6At1g66160 U-box domain-containing protein 1.3 1.6 At5g47960 RASsuperfamily GTP-binding protein 1.3 1.6 (SMG1) At1g49230 RING-H2 fingerprotein RHA3a 1.2 1.5 At4g20000 SigA binding protein family 1.2 1.5

TABLE 2 Sequences of primers used (SEQ ID NOS 2-70, respectively, inorder of appearance) Gene Primer Name Sequence (5′→3′) RT-PCR primers:At3g18780 Actin-FP CTAAGCTCTCAAGATCAAAGGCTTA Actin-RPACTAAAACGCAAAACGAAAGCGGTT Actin2-F GAAGTACAGTGTCTGGATCGGTGGTT Actin2-RATTCCTGGACCTGCCTCATCATACTC At1g71870 At1g71870-FTGTGGTTTGGGTTGCTTTCAGCTC At1g71870-R TCAGTCTCATTGCCTTCACGGCTT At3g05730At3g05730-F ATGGCAAAGACCCTCAATTCCATCTG At3g05730-RTATTTCAACGACCGTAGCAGTGGC At3g16670 At3g16670-FTCCTCAACATAGTCGCTATCCTCCCA At3g16670-R GAGAAGGGAAACACACTGTAACCGAACAt5g10040 At5g10040-F TTGCTGTGGCGGTTTCTAGTGGCTTT At5g10040-RACATGCCCTCTGGTGATTAGAGAAGC At2g02990 At2g02990-FCTGGTTCCGGTTTAATCGAATGTCCG At2g02990-R GATCGATGCCGGTTCAAGAGACTGAAAt2g40960 At2g40960-F AGCTGGTACCATGGACACAGCATTGACC At2g40960-RCCGGGAGCTCTTACCGGTTCTGCATG At2g41230 At2g41230-FCCTCCTCCTTCCTCTACTCCTCATGATT At2g41230-R TTATGTATGTACGGACGGTTCGCAACGCAt5g46295 At5g46295-F TGAGAAGATGATGAGAAAAGGGAAGCTTTC At5g46295-RTGTTAGAATTTACAACCACAACAGAGGAAG At1g50060 At1g50060-FCAGTGAAGATAGGGTGTGCTAGGGTT At1g50060-R ATCAGTAAGGGTACTCTCCGACCCAAAt3g62760 At3g62760-F ATCTCCACCACGTGCCTTACACTTAC At3g62760-RTTAAGGAAAGCCGGACGAGAACAGAG At5g14750 At5g14750-FTGGGTTCATGAGGATGAGTTTGAGC At5g14750-R GACTGTTGATGTATTAGTGTTTGATCAGCAt5g15725 At5g15725-F CGACCAAGGATATAATATGAAGAAGACGAG At5g15725-RGTCAATTAGTGACGATTACGCACGCC At1g74490 At1g74490-FTTTAGTCCTTAGGATGTCTGAGAAACCC At1g74490-R GGTTAGACCATCGATGCTTGAGGTAt4g38080 At4g38080-F GCCCACAATCCCTAACATTCCACAGA At4g38080-RAGTGTGTGATCCAAAGCTGTCTCAGG At1g35210 At1g35210-FGGTTTGGTAATGGGCACAAAGAAGAG At1g35210-R CTTGCACGTACCCACCAAACTGATCTAt1g77640 At1g77640-F CGGAGATCCGTTTGATTATTCTCCAC At1g77640-RTGGACCGTTGGATTAACTGAAACTCC At3g56710 At3g56710-FGTGATTGTTATGAGCCGTTGAATGCGG At3g56710-R TCACATAGAATCGATGCTTCCAAAGTCAAt5g37770 At5g37770-F GTGAGAAGTGCTCTGTGCAAGATTGT At5g37770-RCGGCGAAATCTTCCAAATCCTCAAGC At5g39670 At5g39670-FCGATGGAAGTAAAGACGGAAGAATCG At5g39670-R GGTGCGGAGACAACAGTATTAACAGACAt2g43290 At2g43290-F AGGTGGTGGCTTTAGCAGCAGTA At2g43290-RACACCTTCCTCGATTACACGATGTT At4g25470 At4g25470-FTTGATGTCGAGGGAGATGATGACGTG At4g25479-R ACCATTTACATTCGTTTCTCACAACCAAAt5g49520 At5g49520-F CCTTCGCAGATCAGATCCGATACTATT At5g49520-RACTCCTCATGAAACCTACCTACCGGA At4g11280 At4g11280-FGAAGAAGTGTTGGCAGAGTAACCTCAG At4g11280-R TCTGTGCACGGACTAGCGGAGAATAIL-PCR primers: Degenerate primers: AD1: NTCASTWTWTSGWGTT AD2:NGTCGASWGANAWGAA AD3: WGTGNAGWANCANAGA pSKI015-specific primers:ACT-TAIL1: TGGATTGATGTGATATCTAGATCCG ACT-TAIL2:CCCCCACCCACGAGGAACATCGTGG ACT-TAIL3: GGAAGATGGCTTCTACAAATGCCATPrimers to genotype MTF mutant plants: MTF-RT forward:CTCATCCCTATCTCTCAAACC MTF reverse: TTCCGGCAGGGAAGAGCTTAAGCATCTTT-DNA primer LBa1: TGGTTCACGTAGTGGGCCATCG Primers to amplify MTF cDNA:MTF-XhoI-F ACGGCTCGAGATGAGAGAAGATAATCCA MTF-SpeI-RAACCACTAGTTTAATTTCCGGCAGGGAAG Real-time RT-PCR primers: MTF-RT forwardCTCATCCCTATCTCTCAAACC MTF-RT reverse TCTGAAGATGACTCGCAACGT qORA59-FTCGCGGCCGAGATAAGAGACTC qORA59-R TCCGGAGAGATTCTTCAACGACATCCMTF RNAi primers: MTF-RNAi-F ACACTAGTGGCGCGCCTTTACCTTAGGAGAATGCMTF-RNAi-R ACGGATCCATTTAAATTTGATCCTGACGACAAAT MTF promoter primers:MybPro-AgeI: CCCCACCGGTATACTACAAAATACCTAAAACAAAATGT MybPro-BamHI:CCAAGGATCCGAGATGGAAGCTCTTCTTC

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The invention claimed is:
 1. A method to increase transformationsusceptibility in a plant selected from the group consisting ofBrassica, Arabidopsis, and rice, the method comprising: (a)down-regulating expression of endogenous genes encoding mybtranscription factors in Brassica, Arabidopsis, or rice by using RNAi ortrans-zeatin secretion (TZS) expressing bacteria, wherein the mybtranscription factor in Brassica is selected from the group of aminoacid sequences consisting of SEQ ID NO: 71 encoded by SEQ ID NO: 78, SEQID NO: 72 encoded by SEQ ID NO: 79, and SEQ ID NO: 73 encoded by SEQ IDNO: 80, and combinations thereof; the myb transcription factor inArabidopsis is SEQ ID NO: 74 encoded by SEQ ID NO: 76; and the mybtranscription factor in rice is SEQ ID NO: 75 encoded by SEQ ID NO: 77,and (b) contacting the Brassica, Arabidopsis, or rice plant having thedownregulated myb transcription factor with Agrobacteria, therebyincreasing transformation susceptibility.
 2. A method to increasetransformation susceptibility in a plant selected from the groupconsisting of Brassica, Arabidopsis, and rice, the method comprising:(a) decreasing expression of a gene encoding a functional ortholog of afamily of myb transcription factors designated MTF in Arabidopsis usingRNAi or TZS in Brassica, Arabidopsis, and rice; wherein thetranscription factors negatively regulate plant transformationsusceptibility; and wherein the nucleic acid sequence encoding thefunctional ortholog is selected from the group consisting of SEQ ID NOs:78, 79, 80 and combinations thereof in Brassica; the nucleic acidsequence encoding the functional ortholog is SEQ ID NO: 76 inArabidopsis; and the nucleic acid sequence encoding the functionalortholog is SEQ ID NO: 77 in rice; and (b) contacting the Brassica,Arabidopsis, or rice plant having the downregulated myb transcriptionfactor with Agrobacteria, thereby increasing transformationsusceptibility.